A tissue engineered corneal epithelium replacement for animal testing using human stem cells

The use of human stem cells in the treatment of serious disease or injury is currently undergoing a phase of rapid development. This intensive research has already produced new treatments for blindness. UK and Japanese scientists are now growing adult stem cells taken from donor corneas (the transparent tissue at the front of the eye) on specialised membranes to create new functional tissue. These regenerated tissues are then transplanted onto the surface of diseased or injured eyes leading to a restoration of the patients' sight. The use of human stem cells need not be limited to clinical theraputic use i.e. aimed at re-establishing vision in humans, they can aslo be used to significantly reduce the number of animals used in medical research. Presently, thousands of rabbits are needed every year to test for the toxic potential of new drugs and cosmetics. The method used in testing on rabbits eyes has changed little over 50 years. Now, by employing the scientific advancements recently made in therapeutic stem cell research to the manufacture of a human tissue based replica in culture, we aim to develop a human stem cell based non-animal alternative to drug and nanomaterial testing. The advantages of this are primarily that the effect of new chemicals/materials can be assessed without any animals being harmed and since the replica tissue is formed from human stem cells the results are also more representative. Furthermore, our non-animal model can assess water insoluble materials such as nanomaterials, something the animal models can not do well. To develop a human stem cell based non-animal alternative to drug testing we will essentially recreate the surface of the human eye in a plastic dish. Using adult stem cells isolated from adult corneal donor tissue and building upon the techniques sucessfully employed for corneal stem cell transplantation, we will identify a suitable protocol and substrate capable of both supporting the formation of a functional corneal epithelium in vitro and being easily sourced. The suitability of the newly developed human stem cell based non-animal alternative to drug testing will be established by vigorous testing of the model's response to known toxic and non-toxic agents. The model's response to toxicity testing will be robustly assessed by microscopy and established toxicity assays.

We will apply our knowledge of clinical human corneal stem cell transplantation and cornea/amniotic membrane collagen fibre structure to construct a model specifically designed to make possible an accurate non-animal investigation into the effect chemicals and nanoparticles have on the surface of the eye. Corneal stem cells will be isolated from human donor tissue and expanded upon a specifically engineered collagen sheet. The collagen sheet will manufactured in such a way as to closely resemble the native structure underlying corneal epithelial cells within the eye. The bespoke collagen substrates ability to support ex vivo stem cell expansion and epithelial startification will be compared against a previously established and clinically sucessful substrate for in vitro corneal epithelial cultivation, that being amniotic membrane. Validation of the stem cell based model, as a non-animal alternative for oculotoxicity testing, will be accomplished by comparing the dose-dependent toxic effect of a range of chemicals with known toxicity. Furthermore, the toxicity of novel nanoparticles on ocular surface function will also be investigated. Epithelial toxic damage, to our stem cell based model, will be measured directly by microscopy and indirectly by cell toxicity assays.